TEXT

Nanotechnology has emerged as a revolutionary technique in a wide array of applications (1). Gold nanoparticles (AuNPs), in particular, have been the subject of extensive study for use in biomedical applications, including drug delivery, diagnosis, and therapy. Gold nanoparticles are promising drug delivery carriers due to their small size, high surface area for drug loading, and good cell penetration, and they enhance drug bioavailability and reduce drug resistance by specifically targeting cellular functions (1, 2).

trans-Cinnamic acid (CA) is a natural organic compound that is found in a variety of plants and is a main chemical constituent of cinnamon (Cinnamomum cassia) (3). CA and its derivatives possess pharmacological value due to their broad-spectrum bioactivities, such as anticancer (4), antidiabetic (5), antioxidant (6), antibacterial (7), antifungal (8), antimycobacterial (9), anti-inflammatory (10), and enzyme inhibition activities (11). However, low bioavailability of CA due to its hydrophobic characteristics is a limiting factor in in vivo models. For the first time, we conjugated AuNPs with CA (yielding CA-AuNPs) and tested their effects against the protist pathogen Acanthamoeba castellanii, belonging to the T4 genotype, Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), and Gram-negative neuropathogenic Escherichia coli K1.

Synthesis and characterization of CA-AuNPs.Synthesis of CA-AuNPs was achieved by reduction of tetrachloroauric acid with sodium borohydride in the presence of CA. Briefly, a 1 mM stock solution of CA was prepared in 10% methanol, while tetrachloroauric acid was dissolved in water to obtain a 5 mM stock solution. Next, 1 ml of 1 mM CA was reacted with 15 ml of a 1 mM tetrachloroauric acid aqueous solution, and the reaction mixture was magnetically stirred for 10 min. Next, 30 μl of a freshly prepared 4 mM sodium borohydride aqueous solution was added as a reducing agent to the aforementioned stirred reaction mixture. The color of the solution turned from pale yellow to pink/red on addition of the reducing agent, indicating the reduction of gold ions and the formation of CA-AuNPs. Unprotected bare AuNPs were synthesized by the same procedure but in the absence of any stabilizing agent (12). Synthesized CA-AuNPs were characterized by UV-visible spectrophotometry, dynamic light scattering (DLS), Fourier transform infrared (FT-IR) spectroscopy, and atomic force microscopy (AFM) for determination of stabilization and morphology, as described in our previous studies (13). CA alone showed maximum absorbance at 265 nm in the UV-visible spectrum, while CA-AuNPs gave a characteristic surface plasmon resonance band for AuNPs at 540 nm, which corresponds to successful stabilization of AuNPs by CA (Fig. 1A). For CA-AuNP size and morphological determinations, AFM images were recorded. CA-AuNPs were found to be spherical in shape and polydispersed in size (Fig. 1B). CA-AuNPs fell in the wide size range of 10 to 100 nm, with an average size of 89 nm, as measured by using a DLS Zetasizer, while a zeta potential of −20 mV was recorded. FT-IR spectral analysis of CA-AuNPs showed the chemical modifications involved in the stabilization of AuNPs with CA molecules. CA alone showed absorption bands at 3,418 cm−1 for OH stretching and 1,687 cm−1 for C=C stretching, as reported in the literature (14). After the formation of CA-AuNPs, the OH stretching vibration shifted to 3,327 cm−1, which suggests the interaction of carboxylic acid with gold for the stabilization of AuNPs. CA-AuNPs, CA alone, and AuNPs alone were subsequently tested for antiamoebic and antibacterial properties.

(A) UV-visible spectrum of CA-AuNPs, showing a surface plasmon resonance band at 540 nm, which indicates the successful formation of CA-conjugated AuNPs. (B) AFM image of CA-AuNPs. Extensive imaging was performed to derive a representative image of the morphology of nanoparticles. The average size of CA-AuNPs was found, using DLS analysis, to be 89 nm.

CA-AuNPs exhibited significant amoebicidal effects against A. castellanii.Amoebicidal assays were performed by incubating A. castellanii (5 × 105 parasites) with different concentrations of CA-AuNPs, CA alone, or AuNPs alone, in a final volume of 0.5 ml/well in 24-well plates, as described previously (15). Plates were incubated for 24 h at 30°C. A. castellanii incubated in RPMI 1640 medium alone was used as a negative control, while 40 μM chlorhexidine was used as a positive control. Next, the viability of amoebae was determined by adding 0.1% Trypan blue to each well; live (nonstained) A. castellanii organisms were enumerated using a hemocytometer. The data are expressed as the mean ± standard error of three independent experiments. The results revealed that CA showed moderate effects on A. castellanii viability, compared with the negative control (P < 0.05, using a two-sample t test and two-tailed distribution) (Table 1). Conjugation with AuNPs significantly enhanced the antiamoebic properties of CA (P < 0.05) (Table 1). Conversely, treatment with bare AuNPs alone had no observable adverse effects on amoebae viability (Table 1). These results suggest that CA is a novel antiacanthamoebic agent whose potency is significantly enhanced with AuNP conjugation.

Preparation of A. castellanii cysts and excystation efficacy of CA-AuNPs.A. castellanii cysts were prepared as described previously, through inoculation of 1 × 106 trophozoites on nonnutrient agar plates and incubation at 30°C for up to 14 days (17). Next, each plate was thoroughly washed and scraped with 5 ml of phosphate-buffered saline (PBS), followed by centrifugation at 3,000 × g for 10 min to collect the pellet of cysts. These cysts were then resuspended in PBS, enumerated using a hemocytometer, and used in excystation assays. The effects of CA-AuNPs on excystation were measured by inoculating 5 × 105A. castellanii cysts in PYG medium after treatment with 10 or 5 μM CA-AuNPs, in 24-well plates. CA and AuNPs alone were also used at the same concentrations, as controls. Plates were incubated at 30°C and observed under an inverted microscope every 24 h for up to 72 h to detect the emergence of trophozoites, which were counted using a hemocytometer. The data are expressed as the mean ± standard error of three independent experiments. The results revealed that CA-AuNPs exhibited significant excystation, compared to the negative control (PYG medium), as well as CA alone at both 10 and 5 μM (P < 0.05, using a two-sample t test and two-tailed distribution) (Fig. 2A).

(A) Effects of CA-AuNPs on the excystation of A. castellanii belonging to the T4 genotype. Briefly, 5 × 105A. castellanii cysts were incubated with 10 and 5 μM concentrations of CA-AuNPs in growth medium (PYG medium) at 30°C for 72 h. After this period, amoebae were counted using a hemocytometer. Chlorhexidine (CHX) was used as a positive control, while PYG medium alone was used as a negative control. (B) CA-AuNP blockage of encystation. Briefly, 5 × 105A. castellanii trophozoites were incubated with CA-AuNPs, CA alone, or AuNPs alone at 10 and 5 μM concentrations in encystation medium (E.M) (50 mM MgCl2 and 10% glucose). After 72 h of incubation, 0.5% SDS was added and resistant cysts were enumerated using a hemocytometer. The results represent the mean ± standard error of three independent experiments performed in duplicate. *, P < 0.05, using a two-sample t test and two-tailed distribution.

CA-AuNPs inhibit encystation of A. castellanii.Encystation assays were performed as described previously (17). Briefly, 5 × 105A. castellanii trophozoites were incubated with 10 and 5 μM concentrations of CA-AuNPs, CA alone, or AuNPs alone, in 0.5 ml/well of PBS containing 50 mM MgCl2 and 10% glucose (encystation medium), in 24-well plates. The plates were incubated at 30°C for 72 h and were observed under an inverted microscope every 24 h to detect the formation of cysts. After 72 h, 0.5% SDS was added and mature cysts were enumerated using a hemocytometer. Untreated amoebae suspended in PBS, PYG medium, and encystation medium were used as controls. The data are expressed as the mean ± standard error of three independent experiments. The results revealed that CA-AuNPs blocked encystation significantly, compared to the negative control (encystation medium), as well as CA alone at both 10 and 5 μM (P < 0.05, using a two-sample t test and two-tailed distribution) (Fig. 2B).

Conjugation with AuNPs enhanced the antibacterial effects of CA.Bactericidal assays were performed as described previously (18). Briefly, bacterial cultures were adjusted, using a spectrophotometer, to an optical density at 595 nm of 0.22, corresponding to 108 CFU/ml. Inocula of 10 μl of bacterial culture (equivalent to approximately 106 CFU) were incubated with different concentrations of CA, CA-AuNPs, and AuNPs in 1.5-ml centrifuge tubes, with the final volume adjusted to 0.2 ml. Tubes were incubated at 37°C for 2 h. Bacteria incubated in the solvent alone were used as a negative control, while bacteria incubated with 100 μg/ml gentamicin were used as a positive control. Following this incubation, bacteria were serially diluted and enumerated by plating on nutrient agar plates. The results revealed that CA alone and bare (unconjugated) AuNPs exhibited antibacterial effects against both types of bacteria tested (Table 1). Conjugation of AuNPs with CA significantly enhanced bactericidal efficacy, and bacterial viability was abolished at 5 μM CA-AuNPs (P < 0.05) (Table 1).

CA-AuNPs inhibited pathogen-mediated host cell cytotoxicity.To determine whether CA-AuNPs protected host cells against pathogen-mediated damage, cytotoxicity assays were performed, as described previously (19). Briefly, assays were performed in 96-well plates containing confluent HeLa monolayers. A. castellanii (5 × 105 organisms), MRSA (106 CFU), and E. coli K1 (106 CFU) were incubated with drugs for 2 h at 37°C. Following this incubation, cells were centrifuged at 5,000 × g for 1 min and the supernatant was discarded, to remove extracellular drug. The pellet was resuspended in 200 μl of RPMI 1640 medium and added to the HeLa monolayers to determine cytotoxicity by measuring lactate dehydrogenase (LDH) release. Plates were incubated for 24 h at 37°C in a 5% CO2 incubator. Negative-control values for cytotoxicity assays were obtained by incubating HeLa cells with RPMI 1640 medium alone, and positive-control values were obtained by completely lysing the cells using 1% Triton X-100. After incubation, supernatants were collected, and cytotoxicity was determined using a LDH assay kit (Roche Applied Sciences). The percent cytotoxicity was calculated as follows: % cytotoxicity = (sample absorbance − negative-control absorbance)/(positive-control absorbance − negative-control absorbance) × 100. The findings revealed that A. castellanii, MRSA, and E. coli K1 produced more than 70% host cell cytotoxicity (Fig. 3). Pretreatment with CA-AuNPs significantly inhibited A. castellanii-mediated host cell cytotoxicity (P < 0.05) (Fig. 3A), as well as bacteria-mediated host cell death (Fig. 3B and C). Bare AuNPs alone did not exhibit any significant amoebicidal effects; therefore, it is safe to conclude that AuNP conjugation enhanced CA efficacy. This is likely due to the small size, and thus increased bioavailability, that is the hallmark of nanoparticles. Previous studies suggested that CA likely affects target cells through the generation of reactive oxygen species (6), targeting of the ergosterol pathway (20), and/or better drug-cell interactions (21), as well as interactions with thiolated enzymes (22). Other studies revealed that CA affects the cell cycle, resulting in an increase in the G0/G1 phase and thus inhibiting growth and leading to apoptosis (23), which may explain our findings. Although the precise molecular mechanisms of action of CA are not known, two possible mechanisms are suggested, i.e., CA inhibits the expression of cell proliferation genes and/or CA blocks the posttranslational modification of cell-growth-regulating proteins, such as certain p21ras proteins, thus preventing protein prenylation by inhibiting the synthesis of mevalonate-derived residues (23). These residues bind poorly to the cell membrane, resulting in growth inhibition. However, precise mechanistic studies and assessment of their in vivo potential are needed to determine the clinical value of CA-AuNPs. Recently, various drug-conjugated nanoparticles have been being developed to target resistant microbes (24). The most common metal carriers for nanoparticle-based drug delivery systems include gold, silver, and iron oxide, due to their inertness and biocompatibility (25). Our studies support these findings and clearly show that nanoparticles hold great promise in biomedical applications due to advantages in drug delivery, including increased bioavailability, reduced side effects, and specific and controlled drug release.

(A) CA-AuNP inhibition of A. castellanii-mediated host cell cytotoxicity. Briefly, amoebae were pretreated with 5 μM drugs for 2 h, followed by incubation with host cells to determine host cell death, as described in the text. In the absence of drug, amoebae produced approximately 80% host cell death. At micromolar concentrations, CA-AuNPs significantly inhibited A. castellanii-mediated host cell death (P < 0.05, using a two-sample t test and two-tailed distribution). (B and C) CA-AuNP inhibition of host cell cytotoxicity due to MRSA (B) and E. coli K1 (C) (P < 0.05, using a two-sample t test and two-tailed distribution). Chlorhexidine (CHX) and gentamicin abolished pathogen-mediated host cell cytotoxicity. The data are presented as the mean ± standard error of at least three independent experiments performed in duplicate.

In conclusion, these findings showed that conjugation of CA with AuNPs enhanced the antiacanthamoebic and antibacterial properties of CA. Because nanoparticle-based drug delivery systems are anticipated as a relatively new class of drug delivery systems, these findings provide a step forward in the development of new and effective antimicrobial nanomedicine agents. It is hoped that CA-AuNPs can serve as a potential drug lead for improved therapies, although further studies are needed to understand the mode of action and the in vivo potential of CA-AuNPs.

ACKNOWLEDGMENTS

This work was supported by university research award INT-2017-03 from Sunway University, Malaysia.

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